miR-146a Attenuates Sepsis-Induced Myocardial Dysfunction by Suppressing IRAK1 and TRAF6 via Targeting ErbB4 Expression

Abstract

Myocardial dysfunction is a major manifestation of sepsis and closely associated with the increased mortality. MicroRNA-146 is one of the most important microRNAs identified as a potent negative regulator in innate immune and inflammatory responses induced by lipopolysaccharide (LPS). We aimed to identify the role and potential regulatory mechanism of miR-146a in sepsis-induced cardiac dysfunction with the induction of ErbB4 signaling. H9C2 cells were treated with LPS to induce sepsis, and miR-146a overexpression significantly increased the cell viability, reduced the apoptosis and ROS level, and attenuated the release of proinflammatory cytokines including TNF-α and IL-1β. Levels of ErbB4, p-NF-κB, NF-κB, TRAF6, IRAK1, caspase 3, Bcl-2, and Bax were measured by Western blot. The overexpression of miR-146a significantly increased the ErbB4 expression, decreased the expression of TRAF6, IRAK1, caspase 3, and the phosphorylation level of NF-κB, and also increased the Bcl-2/Bax ratio, suggesting the inhibition of inflammation and apoptosis. The protective effects were all abolished by the use of siErbB4. In conclusion, our results demonstrated that the overexpression of miR-146a mitigates myocardial injury by negatively regulating NF-κB activation and inflammatory cytokine production via targeting ErbB4 in LPS-induced sepsis.

Figure 1

ROS level detected by DHE assay. LPS significantly increased the ROS level.

Figure 2

LPS increased miR-146a expression in cardiomyocytes detected by qPCR. ^∗P < 0.05 versus the control group.

Figure 3

The change of cell viability detected by CCK8. Data were expressed as mean ± SEM. ^∗P < 0.05 versus the control group; ^##P < 0.01 versus the LPS group; ^$P < 0.05 versus the LPS + miR group.

Figure 4

ROS level detected by DHE assay. Overexpression of miR-146a significantly reduced the ROS level post-LPS treatment.

Figure 5

Overexpression of miR-146a downregulated TRAF6 and IRAK1 expression and suppressed NF-κB activity in cardiomyocytes following LPS treatment. Representative images of the Western blot results are shown. The values were expressed as the mean ± SEM. ^∗∗P < 0.01 versus the control group; ^#P < 0.05 versus the LPS group; ^##P < 0.01 versus the LPS group; ^$P < 0.05 versus the LPS + miR group; ^$$P < 0.01 versus the LPS + miR group.

Figure 6

ROS level detected by DHE assay. Overexpression of miR-146a significantly reduced the ROS level post-LPS treatment, and siErbB4 significantly reversed the effect.

Figure 7

Overexpression of miR-146a decreased the levels of inflammatory cytokines in LPS-treated cardiomyocytes. The use of siErbB4 significantly reversed the effect of miR-146a. Data were expressed as mean ± SEM. ^∗∗P < 0.01 versus the control group; ^#P < 0.05 versus the LPS group; ^##P < 0.01 versus the LPS group; ^$P < 0.05 versus the LPS + miR group; ^$$P < 0.01 versus the LPS + miR group.

Figure 8

Evaluation of the apoptosis rate by TUNEL staining. Representative images of apoptosis are shown. The apoptotic cells were detected by TUNEL (green); the nuclei were detected by DAPI (blue). The results were expressed as the mean ± SEM. ^∗∗P < 0.01 versus the control group; ^##P < 0.01 versus the LPS group; ^$$P < 0.01 versus the LPS + miR group.

Figure 9

The effect of overexpression of miR-146a on the expression of ErbB4, TRAF6, IRAK1, NF-κB, and caspase 3 activity and Bcl-2/Bax ratio. Representative images of the Western blot results are shown. The overexpression of miR-146a significantly increased the ErbB4 expression, decreased the expression of TRAF6, IRAK1, caspase 3, and the phosphorylation level of NF-κB, and also increased the Bcl-2/Bax ratio; the effects were reversed by the use of siErbB4. The values were expressed as the mean ± SEM. ^∗P < 0.05 versus the control group; ^∗∗P < 0.01 versus the control group; ^#P < 0.05 versus the LPS group; ^##P < 0.01 versus the LPS group; ^$P < 0.05 versus the LPS + miR group; ^$$P < 0.01 versus the LPS + miR group.